ELECTRON TOMOGRAPHIC STUDY OF HERPES SIMPLEX VIRUS 1 ENVELOPMENT AND EGRESS

单纯疱疹病毒 1 包络和流出的电子断层扫描研究

• 批准号: 7721696
• 负责人: JOEL D. BAINES
• 金额: $ 2.22万
• 依托单位: WADSWORTH CENTER
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-02-01 至 2009-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7721696
• 关键词: 3-Dimensional; ATP Hydrolysis; Accounting; Actins; Biological Preservation; Blindness; Caliber; Candidate Disease Gene; Cell Nucleus; Cell physiology; Cells; Complex; Computer Retrieval of Information on Scientific Projects Database; Condition; Cryoelectron Microscopy; Cytoplasm; Cytoskeletal Proteins; DNA Packaging; Electrons; Encephalitis; Extracellular Space; Extracellular Structure; Fiber; Freezing; Funding; Genes; Goals; Grant; Herpesviridae; Herpesvirus 1; Human; Immune; In Situ; Indium; Individual; Institution; Laboratories; Lamins; Manuscripts; Medical; Membrane; Modeling; Morphologic artifacts; Nature; Nuclear; Nuclear Envelope; Nuclear Inner Membrane; Nuclear Outer Membrane; Nucleocapsid; Numbers; Pathway interactions; Patients; Plastics; Preparation; Process; Production; Proteins; Protocols documentation; Reaction; Recruitment Activity; Reporting; Research; Research Personnel; Resources; Science; Shapes; Simplexvirus; Source; Staging; Structure; Surface; Techniques; Testing; Tomogram; Transport Vesicles; UL34 gene; United States National Institutes of Health; Universities; Viral; Viral Genes; Viral Proteins; Virion; Virus; Virus Assembly; Work; abstracting; animation; electron tomography; experience; extracellular; insight; movie; mutant; novel; particle; pathogen; pressure; reconstruction; sample fixation; terminase; three dimensional structure; tomography; viral DNA; viral envelope lipids

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. SUPPORT: NIH 5R01AI052341-02 "Nucleocapsid envelopment Herpes simplex virus-1", 03/03-02/08 Joel D. Baines Cornell University NIH 2R01GM050740-09 "Herpes Simplex Virus Terminases", 01/95-12/07 Joel D. Baines Cornell University NIH F32GM067519 "ATP hydrolysis in HSV DNA packaging" to C.L. Duffy, 2003-2005. C.L. Duffy Cornell University ABSTRACT: Extracellular herpesvirus virions consist of the nucleocapsid surrounded by an amorphous proteinaceous layer called the tegument that is in turn surrounded by a lipid viral envelope. The latter is derived from host membranes from which virions bud. The particles are known to bud from the inner nuclear membrane where they acquire an initial virion envelope. Enveloped virions then accumulate between the leaflets of the nuclear membrane, and the virion envelope eventually fuses with the outer nuclear membrane leaflet, dumping the de-enveloped nucleocapsid into the cytoplasm, where it eventually becomes re-enveloped and is secreted into the extracellular space. The involvement of possible host cytoskeletal elements in the production of virions can only be studied by the use of electron tomography because tomography allows analysis of individual particles in-situ and without bias due to symmetry-averaging. Preliminary observations have changed the existing paradigm of how the envelopment process occurs, and suggest that herpesviruses exploit the cell in ways that were not predicted. Herpesviruses cause a number of important medical conditions in humans including blindness and encephalitis in both immune-compromised and immune competent patients. The ultimate goal of this study is to understand the mechanisms by which herpesviruses become enveloped at the nuclear membrane and their pathway of egress from the perinuclear space. All undergo the same pathway of virion production and exit, thus the studies will establish the paradigm for this important group of pathogens. The conservation of these steps suggest that it is conceivable that compounds that interfere with this pathway would be effective against all herpesviruses. The characterization of herpesvirus particles using tomography will provide a novel view of the virion egress pathway, and new insight for structure and function of virions and viral proteins. The first aim in this project is to elucidate the structure of wild type and mutant particles as they engage the envelopment machinery at the inner nuclear membrane. The nature of the envelopment machinery at the inner nuclear membrane is unknown, although the Baines laboratory has identified three candidates (genes UL11, UL31 and UL34) as important for the envelopment reaction (Baines and Roizman, 1992; Reynolds et al., 2001). Cells infected with wild type and available mutant viruses lacking genes encoding these proteins will be examined by cryoelectron tomography. Preliminary analyses of wild type nascent virions (those located in the perinuclear space), using fixed material, indicate the presence of novel fibers that bridge the space between the herpes simplex virus (HSV) virion envelope and the nucleocapsid. The deduced mass of these fibers is higher than can be accounted for by any viral gene, suggesting the novel possibility that host proteins are used as a structural component during virus assembly. This would be unprecedented. The diameter of the fibers is consistent with cytoskeletal proteins such as actin or lamins. Although the existence of nuclear actin was controversial, its presence is now widely acknowledged, but remains functionally obscure (Bettinger et al., 2004). The fibers are connected to the nucleocapsid in an asymmetrical fashion, and it will be important to verify that this asymmetry is not an artifact of the fixation process, thus cryoelectron microscopy is essential. To test the logical hypotheses that one or more of the envelopment proteins are involved in recruiting the fibers into virions, analysis of the mutant viruses will be performed. If a given protein is involved in recruitment or formation of the fibers, such fibers should be absent or misshapen in particles located at the inner nuclear membrane of cells infected with the relevant mutant virus. A separate, but related issue is whether tegument proteins are incorporated at this stage of the egress pathway. Current dogma suggests that tegument proteins are incorporated only in the cytoplasm after de-envelopment. The structure of individual particles in the perinuclear space will indicate whether at least a subset of tegument proteins are brought into the virion through interaction with the nucleocapsid or the inner nuclear membrane. The second aim is to compare the structure of extracellular virions with that of nascent virions. The comparison might indicate that certain steps occur in the egress pathway occur that have only been alluded to in the past. For example, the hypothesis that additional proteins are incorporated along with nucleocapsids as they become enveloped in the cytoplasm is a longstanding one, and predicts the presence of additional mass in the tegument of extracellular virions (Gr¿newald et al., 2003) as opposed to nascent virions. Our initial tomography work at the RVBC was with plastic sections of conventionally-processed cells. We saw a specialization of the inner leaflet of the nuclear envelope adjacent to, and conforming to, the icosohedral shape of an internal virus particle. We also saw evidence that the nuclear envelope is the origin of viral "transport vesicles" (manuscript in preparation). However, conventional preparation was inadequate to reliably preserve the finer structural details of the virions and their immediate surroundings. Ultimately, we feel that the "native" preparation afforded by frozen-hydrated sections will be required. While awaiting establishment of protocols to do this work with frozen-hydrated sections, we are continuing our work by using high-pressure frozen, freeze-substituted material. Our experience with high-pressure freezing will help us establish optimal freezing protocols with our material prior to starting the frozen-hydrated section work. Electron tomography is the only technique that can yield 3-D ultrastructural information on individual virus particles in-situ. In order to be confident of the results, the native preservation of frozen-hydrated preparation will be required. The technique of frozen-hydrated sections, developed at the RVBC, will be a great asset for our work. References 1. Baines,J.D. and Roizman,B. (1992). The UL11 gene of herpes simplex virus 1 encodes a function that facilitates nucleocapsid envelopment and egress from cells. J. Virol. 66, 5168-5174. 2. Bettinger,B.T., Gilbert,D.M., and Amberg,D.C. (2004). Actin up in the nucleus. Nat. Rev. Mol. Cell Biol. 5, 410-415. 3. Gr¿newald,K., Desai,P., Winkler,D.C., Heymann,J.B., Belnap,D.M., Baumeister,W., and Steven,A.C. (2003). Three-dimensional structure of herpes simplex virus from cryo-electron tomography. Science 302, 1396-1398. 4. Reynolds,A.E., Ryckman,B., Baines,J.D., Zhou,Y., Liang,L., and Roller,R.J. (2001). UL31 and UL34 proteins of herpes simplex virus type 1 form a complex that accumulates at the nuclear rim and is required for envelopment of nucleocapsids. J. Virol. 75, 8803-8817. In the previous reporting period, eight new double-tilt tomographic reconstructions were made from the high-pressure frozen material, supplementing our previous work with conventionally-prepared material. We studied virus particles just inside the nuclear envelope, between the leaflets of the nuclear membrane, and just outside the membrane at various stages of envelopment, and compared these particles to mature extracellular particles. Surface-rendered models of representative examples of virus particle egress in two stages (just inside the nuclear envelope, and just before egress from the nucleus) were made. Animation movies for presentation were also made. We saw a specialization of the inner leaflet of the nuclear envelope adjacent to, and conforming to, the icosohedral shape of an internal virus particle. We also saw evidence that the nuclear envelope is the origin of viral "transport vesicles". In several tomograms, we caught virus particles just inside the nuclear envelope, between the leaflets of the nuclear membrane, and just outside the membrane at various stages of envelopment, and compared these particles to mature extracellular particles.

该副本是使用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这是调查员的机构。 支持： NIH 5R01AI052341-02“ nucleocapsid Invelope sumperx Virus-1”，03/03-02/08 乔尔·贝恩斯·康奈尔大学 NIH 2R01GM050740-09“单纯疱疹病毒末端”，01/95-12/07 乔尔·贝恩斯·康奈尔大学 NIH F32GM067519“ HSV DNA包装中的ATP水解”到C.L.达菲，2003- 2005年。 C.L.达菲·康奈尔大学 抽象的： 细胞外疱疹病毒病毒由一个被称为团队的无定形蛋白质层围绕的核OCAPSID组成，该层又是由脂质病毒信封围绕的。后者源自病毒芽的宿主膜。已知这些颗粒会从内部核膜上芽芽，在那里它们获得了初始的病毒素包膜。然后，被包膜的病毒体在核膜的传单之间积聚，而病毒粒子包膜最终与外核膜叶片融合在一起，将去发的核OCAPAPID倾倒到细胞质中，最终将其重新发育并分泌到细胞外空间中。可能的宿主细胞骨架元素参与病毒的产生，只能通过使用电子断层扫描来研究，因为断层扫描允许对单位的单位颗粒分析，并且由于平均对称性而没有偏差。初步观察结果改变了现有的范式，即包络过程的发生方式，并表明疱疹病毒以未预测的方式探索了细胞。 疱疹病毒在人类中引起许多重要的医疗状况，包括失明和免疫功能障碍患者的失明和脑炎。这项研究的最终目的是了解疱疹病毒在核膜及其从核周空间中出口的途径所包围的机制。所有这些都经历了病毒粒子生产和退出的相同途径，因此研究将为这一重要的病原体建立范式。这些步骤的保护表明，可以想象的是，对这一途径的干扰将对所有疱疹病毒有效。 使用断层扫描的疱疹病毒颗粒的表征将为病毒式出口途径提供新颖的视野，以及有关病毒体和病毒蛋白的结构和功能的新见解。 该项目的第一个目的是阐明野生型和突变颗粒的结构，它们在内部核膜上参与环境机械。尽管贝恩斯实验室已经确定了三个候选者（ul11，ul31和ul34）对环境反应很重要（Baines and Roizman，1992; Reynolds等，2001，2001）。被冷冻电子断层扫描检查的野生型和缺乏编码这些蛋白质基因的可用突变病毒的细胞将检查。 使用固定材料对野生型新生病毒（位于核周空间中的野生型新生病毒（位于核周空间中的病毒）的初步分析，表明存在新型纤维，这些新型纤维弥合了单纯疱疹病毒（HSV）病毒座和核Ocapsid之间的空间。这些纤维的推导质量比任何病毒基因所能解释的要高，这表明新型的可能性是，宿主蛋白在病毒组装过程中被用作结构成分。这将是前所未有的。纤维的直径与细胞骨架蛋白（如肌动蛋白或层粘连蛋白）一致。尽管核肌动蛋白的存在是有争议的，但现在的存在被广泛认可，但在功能上仍然晦涩难懂（Bettinger等，2004）。纤维以不对称的方式连接到核Ocapsid，并且必须验证这种不对称性不是固定过程的伪像，因此冷冻电子显微镜是必不可少的。为了测试一种或多种包膜蛋白参与将纤维募集到病毒中的逻辑假设，将对突变病毒进行分析。如果给定的蛋白参与募集或形成纤维，则该纤维应在感染相关突变病毒的细胞内部核膜的颗粒中不存在或散落。 一个单独的但相关的问题是，是否在出口途径的这一阶段合并了Tegument蛋白。当前的教条表明，仅在开发后仅在细胞质中掺入tegument蛋白。核周空间中各个颗粒的结构将指示是否至少通过与核Ocapsid或内部核膜相互作用将至少一部分Tegument蛋白带入病毒粒子中。 第二个目的是将细胞外病毒的结构与新生病毒的结构进行比较。比较可能表明，在过去的出口途径中发生了某些步骤。例如，假设在细胞质中包裹时与核ocapsid一起掺入了其他蛋白质，这是一个长期存在的假设，并且预测细胞外病毒团队中存在额外的肿块（Gr�Newald等人，2003年），与新生病毒相反。 我们在RVBC的最初的断层扫描工作是传统加工细胞的塑料部分。我们看到了与内部病毒颗粒的二象形状相邻并符合的核包膜内部小叶的专业化。我们还看到证据表明核包膜是病毒“运输蔬菜”的起源（准备中的手稿）。但是，传统的准备不足以可靠地保留病毒及其周围环境的最终结构细节。最终，我们认为将需要由冷冻水合的部分提供的“本地”准备工作。在等待建立协议以使用冷冻水合的部分进行这项工作的同时，我们正在通过使用高压冷冻，冷冻取代的材料继续工作。我们在高压冻结方面的经验将有助于我们在开始冷冻水合部分工作之前使用材料建立最佳的冷冻协议。 电子断层扫描是唯一可以在原位上获得有关单个病毒颗粒的3-D超微结构信息的技术。为了对结果充满信心，将需要进行冷冻水分准备的本地准备。在RVBC开发的冷冻水合部分的技术将是我们工作的重要资产。 参考 1。Baines，J。D.和Roizman，B。（1992）。单纯疱疹病毒1的UL11基因编码一种促进细胞中核ocapsid包膜和出口的函数。 J. Virol。 66，5168-5174。 2。Bettinger，B.T.，Gilbert，D.M。和Amberg，D.C。（2004）。肌动蛋白在细胞核中。纳特。摩尔牧师。细胞生物。 5，410-415。 3。Gr¿Newald，K.，Desai，P.，Winkler，D.C.，Heymann，J.B.，Belnap，D.M.，Baumeister，W。和Steven，A。C.（2003）。从冷冻电子断层扫描中的单纯疱疹病毒的三维结构。科学302，1396-1398。 4。Reynolds，A.E.，Ryckman，B.，Baines，J.D.，Zhou，Y.，Liang，L。和Roller，R。J.（2001）。单纯疱疹病毒1型的UL31和UL34蛋白形成了一种在核边缘积累的复合物，是核OCAPSID环境所必需的。 J. Virol。 75，8803-8817。 在上一个报告期间，由高压冷冻材料进行了八次新的双倾斜断层扫描重建，并补充了我们以前的常规准备材料的工作。我们研究了核包膜内，核膜叶子之间以及在环境的各个阶段的膜外部的病毒颗粒，并将这些颗粒与成熟的细胞外颗粒进行了比较。在两个阶段（就在核包膜内以及从核用户出口之前），在两个阶段（就在核用户出口之前）的表面渲染模型。还制作了用于演示的动画电影。 我们看到了与内部病毒颗粒的二象形状相邻并符合的核包膜内部小叶的专业化。我们还看到了证据表明核包膜是病毒“运输蔬菜”的起源。在几个断层图中，我们在核膜的传单之间以及膜外部的各个阶段在核膜的传单之间捕获了病毒颗粒，并将这些颗粒与成熟的细胞外颗粒进行了比较。